A. Gibberellins
Gibberellins (GAs) are a family of diterpenoid plant growth hormones some of which are bioactive growth regulators. GAs are required for controlling such diverse processes as seed germination, cell elongation and division, leaf expansion, stem elongation, flowering, and fruit set. GAs have been the subject of many physiological, and biochemical studies, and a variety of plant mutants with altered patterns of GA biosynthesis or response have been studied (Graebe, J. E., Ann. Rev. Plant Physiol. 38:419-465 (1987)). However, none of the genes involved in GA synthesis have yet been cloned.
Extensive biochemical studies on endogenous GA intermediates in GA-responsive dwarf mutants have allowed the determination of the GA biosynthetic pathway and several genetic loci involved in GA biosynthesis (reviewed by Graebe, J. E., Ann. Rev. Plant Physiol. 38:419-465 (1987)). A number of the GA responsive dwarf mutants have been isolated from various plant species, such as maize, pea, and Arabidopsis (Phinney, B. O. et al., "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant Growth Substance, ed., P. F. Waering, New York: Academic (1982) pp. 101-110; Ingram, T. J. et al., Planta 160:455-463 (1984); Koornneef, M., Arabidopsis Inf. Serv. 15:17-20. (1978)). The dwarf mutants of maize (dwarf-1, dwarf-2, dwarf-3, dwarf-5) have been used to characterize the maize GA biosynthesis pathway by determining specific steps leading to biologically important metabolites (Phinney, B. O. et al., "Chemical Genetics and the Gibberellin Pathway" in Zea mays L. in Plant Growth Substance, ed., P. F. Waering, New York: Academic (1982) pp. 101-110; Fujioka, S. et al., Plant Physiol. 88:1367-1372 (1988)). Similar studies have been done with the dwarf mutants from pea (Pisum sativum L.) (Ingram, T. J. et al., Planta 160:455-463 (1984)). GA deficient mutants have also been isolated from Arabidopsis (ga1, ga2, ga3, ga4, ga5) (Koornneef, M., et al., Theor. Appl. Genet. 58:257-263 (1980)). One of the most extensive genetic studies of GA mutants has been carried out by Koornneef et al. (Theor. Appl. Genet. 58:257-263 (1980); Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)) in the small crucifer, Arabidopsis thaliana. Using ethylmethanesulfonate (EMS) and fast neutron mutagenesis, Koornneef has isolated nine alleles mapping to the GA1 locus of A. thaliana (Koornneef et al. (Theor. Appl. Genet. 58:257-263 (1980); Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)).
A. thaliana ga1 mutants are non-germinating, GA-responsive, male-sterile dwarfs, whose phenotype can be converted to wild-type by repeated application of GA (Koornneef and van der Veen, Theor. Appl. Genet. 58:257-263 (1980)). Koornneef et al. used three independent alleles generated by fast neutron bombardment (31.89, 29.9 and 6.59) and six independent alleles (NG4, NG5, d69, A428, d352 and Bo27) generated by ethyl methane sulfonate mutagenesis to construct a fine-structure genetic map of the A. thaliana GA1 locus (FIG. 2A). One of the fast-neutron-generated mutants, 31.89, failed to recombine with the six alleles indicated in FIG. 2A, and was classified as an intragenic deletion (Koornneef et al., Genet. Res. Camb. 41:57-68 (1983)).
The ga1 mutants contain reduced levels of GAs and the ent-kaurene synthetase activity in cell-free preparations from ga1 mutants is very low compared to wild type (Barendse et al., Physiol. Plant. 67: 315-319 (1986); Barendse and Koornneef, Arab. Inf. Serv. 19: 25-28 (1982)). Zeevaart, Plant Research '86, Annual Report of the MSU-DOE Plant Research Laboratory, (East Lansing, Mich.), pp. 130-131 (1986), reported that application of ent-kaurene also restored growth of the ga1 mutants, and that .sup.14 C-ent-kaurene was metabolized to GAs when applied to the leaves of these mutants. These results suggest that GA biosynthesis in the ga1 mutants is blocked prior to the formation of ent-kaurene, but the rest of the pathway is unaffected by the mutation. Since the ga1 mutants produce chlorophylls and carotenoids, it is unlikely that the mutation affects the synthesis of geranylgeranyl pyrophosphate (GGPP). Therefore, the GA1 locus is probably involved in the conversion of GGPP to ent-kaurene, encoding one of the ent-kaurene synthetases or a regulator needed for formation of the active enzyme. The GA1 locus has been isolated by genomic subtraction (Sun et al., Plant Cell 4:119-128 (1992)).
The enzyme encoded by the GA1 gene is involved in the conversion of GGPP to ent-kaurene (Barendse and Koornneef, Arabidopsis Inf. Serv. 19:25-28 (1982); Barendse et al., Physiol. Plant. 67:315-319 (1986); Zeevaart, J. A. D., in Plant Research '86, Annual Report of the MSU-DOE Plant Research Laboratory, 130-131 (East Lansing, Mich., 1986)), a key intermediate in the biosynthesis of GAs (Graebe, J. E., Ann. Rev. Plant Physiol. 38:419-465 (1987)). ##STR1## Ent-kaurene synthetase has only been partially purified from a variety of plants (Duncan, Plant Physiol. 68:1128-1134 (1981)).
The synthesis of GGPP from mevalonate is common to terpenes. GGPP is a branch point metabolite which is not only the precursor of GAs, but also a precursor of other diterpenes, such as the phytol chain of chlorophylls, and tetraterpenes, such as the carotenoids. The first committed step of the GA pathway is the conversion of GGPP to ent-kaurene in a two-step cyclization reaction. GGPP is partially cyclized to the intermediate, copalyl pyrophosphate (CPP), by ent-kaurene synthetases A and CPP is immediately converted to ent-kaurene by ent-kaurene synthetase B. Since ent-kaurene is a key intermediate in the GA pathway, its synthesis is likely to be a regulatory point for GA biosynthesis. Indeed, ent-kaurene production has been shown to be altered by changes in photoperiod, temperature, and growth potential of tissues in certain species (Chung and Coolbaugh, 1986; Moore and Moore, 1991; Zeevaart and Gage, 1993).
By examining the molecular lesions in several ga1 alleles, a direct correlation of the genetic and physical maps of the GA1 locus was established and a recombination rate of 10.sup.-5 cM per nucleotide was determined for this region of the A. thaliana genome. (Koornneef, Genet. Res. Comb. 41:57-68 (1983)).
The difficulty associated with cloning the GA1 gene and other genes involved in GA biosynthesis has most been likely caused by the unavailability of efficient transformation/selection systems as well as the lack of available protein sequences. Although ga1 mutants have been available for some time, the cloning of the GA1 gene has remained elusive. The claimed invention solves, inter alia, this problem.
B. Gene Cloning
The ability to identify and clone a particular, desired gene sequence from a virus, prokaryote or eukaryote is of tremendous significance to molecular biology. Such cloned gene sequences can be used to express a desired gene product and therefore can potentially be used for applications ranging from catalysis to gene replacement.
A variety of methods have been developed for isolating and cloning desired gene sequences. Early methods permitted only the identification and isolation of gene sequences that possessed a unique property such as proximity to a prophage integration site, capacity for self-replication, distinctive molecular weight, extreme abundance, etc. (The Bacteriophage Lambda, A. D. Hershey, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1971); Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972); Molecular Biology of the Gene, Watson, J. D. et al., (4th ed.) Benjamin/Cummings, Menlo Park, Calif. (1987); Darnell, J. et al. Molecular Biology, Scientific American Books, New York, N.Y. (1986)). Because these methods relied upon distinctive properties of a gene sequence, they were largely (or completely) unsuitable for identifying and cloning most gene sequences.
In order to identify desired gene sequences that lacked a distinctive property, well characterized genetic systems (such as Escherichia coli, Saccharomyces cerevisiae, maize, mammalian cells, etc.) have been exploited. In accordance with this methodology, cells are mutagenized by chemicals, such as UV light, hydroxylamine, etc. (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)), or by genetic means, such as transposon tagging (Davis, R. W. et al. A Manual for Genetic Engineering, Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1980)), to produce mutants having discernible genetic deficiencies. A desired gene sequence is then identified by its capacity to complement (i.e. remedy) the genetic deficiencies of such mutant cells. Such genetic identification permitted the genetic characterization of the gene sequences, and the construction of genetic maps which localized the gene sequence to a region of a particular chromosome (Taylor, Bacteriol. Rev. 34:155 (1970)). With the advent of recombinant DNA technologies, it became possible to clone (i.e. to physically isolate) such genetically characterized gene sequences. Random fragments of a genome could be introduced into self-replicating vectors to produce gene libraries, each of whose members contain a unique DNA fragment (Maniatis, T. et al., In: Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982)). By screening the members of such libraries for those capable of complementing the deficiency of a mutant cell, it was possible to clone the desired gene sequence.
Although these methods permit the identification and cloning of many gene sequences, they may be employed only where a host cell exists that has a mutation conferring a discernible deficiency, and the gene sequence can be cloned into a gene sequence delivery system (such as a vector) capable of entering the host cell and being expressed.
The capacity to physically isolate certain gene sequences has led to the development of methods that are capable of isolating a desired gene sequence even in the absence of mutations or vectors.
In one such technique, known as "chromosome walking," a desired sequence can be obtained by isolating a gene sequence that is capable of hybridizing to a particular reference sequence. This isolated gene sequence is then employed as a reference sequence in a subsequent hybridization experiment in order to clone a gene sequence that is adjacent to, and that partially overlaps, the originally isolated sequence. This newly isolated sequence will be physically closer to the desired gene sequence than was the originally isolated sequence. This process is repeated until the desired gene sequence has been obtained. As will be appreciated, the ability to clone a gene sequence, in the absence of genetic mutants or vectors, requires some initial information concerning the nucleotide sequence or restriction endonuclease digestion profile of the desired sequence.
Alternatively, the chromosome of a virus or cell can be characterized to produce a physical map based on either nucleotide sequence or restriction endonuclease cleavage data (i.e. an RFLP map). Using such a map, restriction fragments of the chromosome can be cloned without any prior determination as to their genetic function.
More recently, gene cloning has been achieved by synthesizing oligonucleotide molecules using sequences deduced from the amino acid sequence of an isolated protein. cDNA copies of isolated RNA transcripts are made and differential colony or library subtractive hybridizations using either two different RNA sources, or cDNA and RNA is performed to identify the desired clone.
Although these methods may be employed even in the absence of mutants or a gene sequence delivery system, they permit a desired gene sequence to be identified and cloned only if sequences naturally linked to the desired sequence have been characterized and isolated, or if the sequence or restriction map of such sequences has been obtained. Since such data are often unavailable, these methods are often incapable of use in identifying and cloning a desired gene sequence.
Two general approaches have been described for cloning sequences that are present in one strain and absent in another. The first approach, differential screening, has been used to clone the esc gene from Drosophila. Using genomic DNA from strains with and without a deletion to probe replicas of a genomic library poses technical difficulties that become daunting for large genomes. In addition, the deletion must cover at least one entire insert in a genomic library that does not contain any repeated sequences.
The second approach, competitive hybridization, provides an alternative to differential screening. This technique was used by Lamar et al. (Cell 37:171-177 (1984)) to isolate clones specific for the human Y chromosome. In accordance with this method, an excess of sheared DNA from a human female is denatured and reannealed along with a small amount of DNA from a male (the male-derived DNA having been previously treated to have sticky ends). Most of the male DNA reassociated with the sheared DNA yielding unclonable fragments lacking sticky ends. Fragments unique to the Y chromosome, however, could only reassociate with the complementary restricted strand (derived from the Y chromosome). Such reassociation thus formed clonable fragments with sticky ends. This technique has also been used successfully to clone DNA corresponding to deletions in the Duchenne muscular dystrophy locus, and choroideremia.
Unfortunately, the competitive hybridization method does not provide a large enough degree of enrichment. For example, enrichments of about one hundred fold were obtained for the sequences of interest in the above experiments. With enrichments of such low magnitude, the technique is practical only when dealing with large deletions. Indeed, even if the deletion covered 0. 1% of the genome, many putative positive clones have to be tested individually by labeling and probing genomic Southern blots (Southern, J., J. Molec. Biol. 98:503-517 (1975)). The method as it stands, then, is not practical for deletions on the order of 1 kbp (kilobasepair) unless one is dealing with a small prokaryotic genome.
Thus, in summary, the ability to clone DNA corresponding to a locus defined only by a mutation is a relatively simply matter when working with E. coli, S. cerevisiae or other organisms in which transformation and complementation with genomic libraries is feasible. Chromosome walking techniques may be used in other organisms to clone genetically defined loci if the mutant was obtained by transposon tagging, if the locus can be linked to markers in an RFLP map, or if an ordered library for the genome exists. Unfortunately, there are numerous organisms in which mutants with interesting phenotypes have been isolated but for which such procedures have not been developed, such as the GA synthesis mutants of A. thaliana. Thus, many gene sequences cannot be isolated using the above methods.
C. Transgenic and Chimeric Plants
Recent advances in recombinant DNA and genetic technologies have made it possible to introduce and express a desired gene sequence in a recipient plant. Through the use of such methods, plants have been engineered to contain gene sequences that are not normally or naturally present in an unaltered plant. In addition, these techniques have been used to produce plants which exhibit altered expression of naturally present gene sequences.
The plants produced through the use of these methods are known as either "chimeric" or "transgenic" plants. In a "chimeric" plant, only some of the plant's cells contain and express the introduced gene sequence, whereas other cells remain unaltered. In contrast, all of the cells of a "transgenic" plant contain the introduced gene sequence.
Transgenic plants generally are generated from a transformed single plant cell. Many genera of plants have been regenerated from a single cell. (Friedt, W. et al. Prog. Botany 49:192-215 (1987); Brunold, C. et al., Molec. Gen. Genet. 208:469-473 (1987); Durand, J. et al., Plant Sci. 62:263-272 (1989) which references are incorporated herein by reference).
Several methods have been developed to deliver and express a foreign gene into a plant cell. These include engineered Ti plasmids from the soil bacterium A. tumefaciens (Czako, M. et al., Plant Mol. Biol. 6:101-109 (1986); Jones, J. D. G. et al., EMBO J. 4:2411-2418 (1985), engineered plant viruses such as the cauliflower mosaic virus (Shah, D. M. et al., Science 233:478-481 (1986)); Shewmaker, C. K. et al., Virol. 140:281-288 (1985)), microinjection of gene sequences into a plant cell (Crossway, A. et al., Molec. Gen. Genet. 202:179-185 (1986); Potrykus, I. et al., Molec. Gen. Genet. 199:169-177 (1985)), electroporation (Fromm, M. E. et al., Nature 319:791-793 (1986); Morikawa, H. et al., Gene 41:121-124 (1986)), and DNA coated particle acceleration (Bolik, M. et al. Protoplasma 162:61-68 (1991)).
The application of the technologies for the creation of transgenic and chimeric plants has the potential to produce plants that cannot be generated using classical genetics. For example, chimeric and transgenic plants have substantial use as probes of natural gene expression. When applied to food crops, the technologies have the potential of yielding improved food, fiber, etc.
Chimeric and transgenic plants having a specific temporal and spatial pattern of expression of the introduced gene sequence can be generated. The expression of an introduced gene sequence can be controlled through the selection of regulatory sequences to direct transcription and or translation in a temporal or spatial fashion.